Articular cartilage has a limited intrinsic ability to heal. For this reason, orthopaedic management of these lesions remains a persistent problem for the orthopedist and patient. The importance of treating injury to articular cartilage is underscored by the fact that several million people are affected in the United States alone by cartilage damage. (See Praemer A, Furner S. Rice D P. Musculoskeletal conditions in the United States: American Academy of Orthopaedic Surgeons; 1999 p. 34-9). Focal lesions of articular cartilage can progress to more widespread cartilage destruction and arthritis that is disabling. Thus, numerous procedures have been developed in an attempt to treat these lesions and halt or slow the progression to diffuse arthritic changes. (See Browne J E, Branch T P. Surgical alternatives for treatment of articular cartilage lesions. J Am Acad Orthop Surg 2000; 8(3):180-9). Surgical procedures to restore articular cartilage include marrow stimulation techniques, autologous chondrocyte transplant (See Browne J E, Anderson A F, Arciero R, Mandelbaum B, Moseley J B, Micheli L J, et al. Clinical outcome of autologous chondrocyte implantation at 5 years in US subjects. Clinical Orthopaedics and Related Research 2005; 436:237-45), and osteoarticular transfer (See Magnussen R A, Dunn W R, Carey J L, Spindler K P. Treatment of focal articular cartilage defects in the knee: a systematic review. Clinical Orthopaedics and Related Research 2008; 466(4):952-62). At present, none of these techniques have been able to restore a normal cartilaginous surface and have suffered from poor integration with the surrounding normal articular cartilage. Frequently, the repair tissue has inferior biochemical and biomechanical properties. The tissue engineering concepts described herein may eliminate many of the problems associated with the current surgical options.
An alternative cell source demonstrating promise for cartilage repair is the adult stem cell. Mesenchymal stem cells (MSCs) are multipotent cells that are capable of differentiating along several lineage pathways. (See Pittenger M F, Mackay A M, Beck S C, Jaiswal R K, Douglas R, Mosca J D, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 284:143-7). From a small bone marrow aspirate obtained from adults, MSCs can be isolated and expanded into billions of cells due to their proliferative capacity. (See Friedenstein A, Chailakhyan R, Gerasimov U V. Bone Marrow Osteogenic Stem Cells: In Vitro Cultivation and Transplantation in Diffusion Chambers. Cell Tissue Kinet 1987; 20(3):263-72). Additional characterization has also identified a panel of immunophenotypic and cell surface markers characteristic of the MSC. (See Haynesworth S, Baber M, Caplan A. Cell Surface Antigens on Human Marrow-Derived Mesenchymal Stem Cells are Detected by Monoclonal Antibodies. J Cell Physiol 1992; 138:8-16).
In vitro and in vivo analyses have demonstrated that culture expanded MSCs can differentiate into osteoblasts, chondrocytes, adipocytes, tenocytes, myoblasts, and neural cell lineages. MSC populations that had been taken out to 15 passages as well as cyropreserved still have the capacity to differentiate and proliferate, suggesting that MSCs may be valuable as a readily available and abundant source of cells in the tissue engineering field. (See Jaiswal N, Haynesworth S E, Caplan A I, Bruder S P. Osteogenic differentiation of purified culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem 1997; 64:295-312; See also Kadiyala S, Jaiswal N, Bruder S P. Culture-expanded, bone marrow-derived mesenchymal stem cells can regenerate a critical-sized segmental bone defect. Tissue Engineering 1997; 3(2):173-85; See also Rickard D J, Sullivan T A, Shenker B J, Leboy P S, Kazhdan I. Induction of rapid osteoblast differentiation in rat bone marrow stromal cell cultures by dexamethason and BMP-2. Dev Bio 1994; 161:218-28). Furthermore, recent studies have demonstrated that the use of allogeneic MSCs can successfully repair bone and other tissue types in various animal models without provoking an adverse immune response. (See Livingston T L, Peter S P, Archambault M, Van Den Bos C, Gorden S, Kraus K, et al. Allogeneic stem cells regenerate a critically-sized canine segmental gap. Journal of Bone and Joint Surgery American 2003; 85-A(10):1927-35; See also Chamberlain G, Fox J, Ashton B, Middleton J. Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing. Stem Cells 2007; 25(11):2739-49). An allogeneic MSC approach provides an off-the-shelf therapy, where allogeneic MSCs are used as universal cells and in turn, provide cells to a much larger clinical population. They are also currently in clinical trials for various disorders or conditions, including cartilage repair, as an allogeneic cell source.
In recent clinical trial results, 30% of patients receiving direct injection of MSCs demonstrated improvement in cartilage and joint condition. (See Chondrogen clinical trial information for the treatment of knee injuries. Osiris Therapeutics, Inc. 2008). Concerns, however, are the long term efficacy of MSCs for cartilage repair. It has been well documented that MSCs during chondrogenesis exhibit mixed phenotypes as opposed to the hyaline phenotype typically displayed by chondrocytes. (See Karlsson C, Brantsing C, Svensson T, Brisby H, Asp J, Tallheden T, et al. Differentiation of human mesenchymal stem cells and articular chondrocytes: analysis of chondrogenic potential and expression pattern of differentiation-related transcription factors. Journal of Orthopaedic Research 2007; 25:152-63). In culture, it has also been reported that MSCs during chondrogenesis express chondrocyte hypertrophy-associated genes, including type X collagen, alkaline phosphatase, matrix metalloproteinase 13, vascular endothelial growth factor (VEGF), and parathyroid hormone-related protein receptor (PTHrPR). (See Mueller M B, Tuan R S. Functional characterization of hypertrophy in chondrogenesis of human mesenchymal stem cells. Arthritis and Rheumatism 2008; 58(5):1377-88). This suggests that MSCs undergoing chondrogenic differentiation may proceed toward the chondrocyte hypertrophy stage, which is typical of endochondral ossification during skeletal development.
Moreover, in in vivo ectopic studies, human MSCs undergoing chondrogenesis can exhibit chondrocyte hypertrophy (typically seen in osteoarthritis) leading to vascular invasion and mineralization. (See Pelttari K, Winter A, Steck E, Goetzke K, Hennig T, Ochs B G. Premature induction of hypertrophy during in vitro chondrogenesis of human mesenchymal stem cells correlates with calcification and vascular invasion after ectopic transplantation in SCID mice. Arthritis and Rheumatism 2006; 54:3254-66). This phenomenon is thus a concern for the clinical application of MSCs in articular cartilage repair, because chondrocyte hypertrophy in neocartilage could ultimately lead to apoptosis, vascular invasion, and ossification, as observed in the cartilage growth plate.
In the body, adult stem cells are often localized to specific chemically and topologically complex microenvironments, or so-called “niches”. Increasing experimental evidence supports the notion that stem cells can adjust their properties according to their surroundings, and select specific lineages according to the cues they receive from their niche. (See Xie L, Spradling A C. A niche maintaining germ line stem cells in the Drosophila ovary. Science 2000; 290(5490):328; See also Fuchs E, Segre J. Stem cells: a new lease on life. Cell 2000; 100:143-55; See also Watt F M, Hogan B L M. Out of eden: stem cells and their niches. Science 2000; 287(5457):1427). To maximize successful stem cell therapy in the repair of a specific tissue type, the microenvironment of the cells should be designed to relay the appropriate chemical and physical signals to them. Mimicking characteristics of the microenvironment during cartilage development is a viable approach. During cartilage development, one of the earliest events is pre-cartilage mesenchymal cell aggregation and condensation resulting from cell-cell interaction, which is mediated by both cell-cell (neural cadherin and neural cell adhesion molecule) and cell-matrix adhesion (fibronectin, proteoglycans, hyaluronic acid and collagens). (See DeLise A M, Fischer L, Tuan R S. Cellular interactions and signaling in cartilage development. Osteoarthritis and Cartilage 2000; 8:309-34). Type I collagen being the predominant matrix protein present in the early stages of development is later transformed to Type II collagen by increased cell synthesis during differentiation. (See Safronova E E, Borisova N V, Mezentseva S V, Krasnopol'skaya K D. Characteristics of the macromolecular components of the extracellular matrix in human hyaline cartilage at different stages of ontogenesis. Biomedical Science 1991; 2:162-8). Multiple growth factors and morphogens such as Wnts, transforming growth factor-beta, and fibroblast growth factors may also be present to support, promote and/or contribute to the regulation of the differentiation process. The present invention extends these findings by, in part, combining MSCs at relatively high cell densities with scaffolds that provide appropriate cues similar to the native extracellular matrix during development.
One way for a biodegradable scaffold to be successful is to make the material's rate of degradation commeserate with the growth of new cartilage and related tissue. Ideally, the scaffold degrades at a rate to substantially maintain structural support during the initial stages of cartilage formation, but also allows space for continuous growth of new cartilage and related tissue.
It is therefore of great importance to develop a scaffold that will overcome these issues and provide the appropriate cues to support chondrogenesis of the stem cells, e.g., MSCs.